Process for manufacturing of tagatose

ABSTRACT

Tagatose is manufactured by hydrolyzing lactose to galactose and glucose and isomerizing galactose to tagatose and chromatographic separation and recycling any unconverted compounds. Thereby high yields of pure tagatose are obtained.

TECHNICAL FIELD OF THE INVENTION

[0001] The present invention concerns enzymatic manufacturing oftagatose, especially D-tagatose.

BACKGROUND OF THE INVENTION

[0002] D-Tagatose is a multi-purpose low-calorie bulk sweetener havingnon-cariogenic and prebiotic properties. D-Tagatose can be used in foodand functional food as well as in pharmaceuticals, cf. U.S. Pat. No.4,786,722, U.S. Pat. No. 5,356,879 and U.S. Pat. No. 5,447,917.

[0003] According to U.S. Pat. No. 5,002,612 and U.S. Pat. No. 5,078,796D-tagatose has been manufactured by hydrolyzing lactose or a lactosecontaining material to a mixture of galactose and glucose using alactase, optionally removing glucose followed by chemical isomerizationof galactose to tagatose.

[0004] U.S. Pat. No. 6,057,135 describes manufacturing of D-tagatosefrom cheese whey or milk, which is hydrolyzed to prepare a mixture ofgalactose and glucose. Glucose is separated from the galactose byfermentation of glucose and subjected to isomerization using L-arabinoseisomerase.

SUMMARY OF THE INVENTION

[0005] In a first aspect of the invention a process is provided formanufacturing of tagatose comprising a) hydrolyzing lactose or a lactosecontaining starting material to obtain galactose and glucose, b)isomerizing the obtained galactose with a L-arabinose isomerase, and c)chromatographic separation of products and unconverted compounds andrecycling of unconverted compounds to the process.

[0006] In a second aspect of the invention a process is provided,wherein steps a) and b) are performed in one reactor.

[0007] In a further aspect of the invention a process is provided formanufacturing of D-tagatose.

[0008] In a still further aspect of the invention a process is provided,wherein the L-arabinose isomerase used in step b) is thermophilic.

[0009] In a still further aspect of the invention a process is provided,wherein the lactase used in step a) is thermophilic.

[0010] In a still further aspect of the invention a process is provided,wherein the temperature used in step(s) a) and/or b) is/are 40-90° C.

[0011] Still further aspects of the invention are given in the appendedclaims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0012]FIG. 1 shows the process of example 1 schematically,

[0013]FIG. 2 shows the process of example 2 schematically, and

[0014]FIG. 3 shows the process of example 3 schematically.

[0015]FIG. 4 shows the result of example 4.

DETAILED DESCRIPTION OF THE INVENTION

[0016] It has surprisingly been found that it is possible to usechromatographic separation and recycling in an enzymatic process formanufacturing of tagatose. Thereby higher yields and a cleaner processare achieved. The total yield as well as the yield for each cycle arehigher.

[0017] Using this process it is possible to recycle and use anyunconverted lactose and galactose. It is also possible to separate theproducts, tagatose and any by-products, especially glucose fromgalactose and use the glucose for other purposes.

[0018] The starting material can be any lactose or lactose containingmaterial.

[0019] Lactose is a by-product from the cheese manufacture. This processgives an opportunity to convert lactose, a low-value product produced inexcess, into a high value product with properties beneficial to humans.This is a way to use lactose. Opportunities for the utilization oflactose have been sought for a long period of time.

[0020] The process involves enzymatic hydrolysis of lactose, enzymaticconversion of galactose into tagatose and optionally chromatographicseparation with recycling of non-converted products.

[0021] The consumption of chemicals etc is low.

[0022] The production of bi-products is low. Only one by-product isproduced, viz. glucose as a glucose syrup/powder, which can be used forfood.

[0023] It is possible to perform the entire reaction in one reactorcontaining enzymes for the hydrolysis of lactose as well as theisomerization of galactose. Thereby step a) and step b) are combined.

[0024] In so doing the complete process is improved in respect of yieldper time unit. Galactose is continuously removed from the reactor byisomerization thereof to tagatose, thus reducing the concentration ofgalactose that would otherwise impede lactase and result in restraint ofthe transformation of lactose to galactose and glucose. It is possibleto carry out the process at high concentration (Brix) because of thehigh process temperature. Evaporation capacity will be saved which againwill result in improved economy as regards investment and working. Theincreased sugar concentration moreover has the effect that the use ofpreservatives can be reduced. Also the first chromatographic separationwill become superfluous which again means improved economy as regardsinvestment and running.

[0025] Especially an enzymatic conversion of lactose to glucose andgalactose with subsequent isomerization of the galactose to tagatose inthe same enzyme reactor has been demonstrated. The initial testsconfirmed that LacS lactase enzyme and L-arabinose isomerase from T.mathranii could function under identical metal ion, buffer andtemperature conditions. The principle was then tested by incubation ofan 800 mM lactose solution (28%) with immobilized lactase andimmobilized isomerase. Samples were analyzed for contents of lactose,glucose, galactose and tagatose by HPLC. During 24 hours of incubationthe concentration of glucose increased to about 800 mM indicating thatall lactose was cleaved. The concentrations of galactose and tagatoseboth increased linearly to about 300 mM. Consequently the degree ofconversion was (300/800)×100%=38%.

[0026] The further enzyme can be introduced into the same reactor, aso-called combi-reactor.

[0027] The tagatose is especially D-tagatose, which is in high demand inthe food industry.

[0028] All lactases can be used in step a). Examples are enzymes derivedfrom the group consisting of Bacillus, Sulfolobus, Thermoanaerobacter,Thermus and Pyrococcus.

[0029] All L-arabinose isomerases can be used in step b). Examples areenzymes derived from the group consisting of Bacillus, Sulfolobus,Thermoanaerobacter and Thermotoga.

[0030] The enzymes can be used in any form. For example is it possibleto use immobilized enzymes.

[0031] Biotechnological Institute, Denmark, can deliver usable lactasesand L-arabinose-isomerases.

[0032] Biotechnological Institute, Denmark found and tested an enzymederived from Thermoanaerobacter mathranii, Thermoanaerobacter mathraniiDSM 11426. They have filed U.S. patent application Ser. No. 09/905,108covering their invention.

[0033] K_(m) values for T. mathranii enzyme on D-galactose are lowerthan values for enzymes from common L-arabinose isomerase producingorganisms, such as Aerobacter aerogenes, Bacillus amyloliquefaciens,Arthrobacter sp, and Lactobacillus pentosus. Therefore T. mathranii hasa better affinity.

[0034] This last mentioned enzyme is thermophilic.

[0035] An added benefit of using thermophilic enzymes is the possibilityof using high process temperature where the solubility of lactose andglucose is higher. This means that more concentrated products can beused for the enzymatic process of the invention. This again means a lesswater consumption and less water for evaporation. This will givetechnical advantages and less need for water and energy i.a. for heatingand cooling process streams.

[0036] The use of thermophilic enzymes has further made it possible towork at a higher temperature. This leads to a better hygiene because ofreduced risk of contamination with damaging microorganisms. Furthermorean increased conversion of galactose to tagatose is achieved at highertemperatures compared to the conversion of arabinose to ribulose. Inaddition hereto there may also be technical advantages such as easierflow and quicker filtration.

[0037] It is thus preferred to use enzymes having optimal yields at hightemperatures in steps a) and/or in step b). This will normally give afaster reaction. Further, it is possible to clean the system using hightemperatures, for example pasteurization temperatures usually used inthe dairy industry or temperatures over 100° C., if the enzymes arethermophilic or even extremophilic. If there is no or only minortemperature difference between the temperature in steps a) and b), theenergy for cooling and heating is reduced. The production process canthus be run at temperatures above 60° C. This has wide implications forthe microbiology and the consumption of steam and brine for warming andcooling.

[0038] The temperature used in step(s) a) and/or b) is/are 40-90° C.Normally it is 60-90° C. Preferably the temperature is 60-80° C., morepreferably 65-70° C., and the most preferred temperature is 65° C.However, the temperature will depend of the chosen enzymes, and it canbe different in steps a) and b). As mentioned above, some advantages areachieved by using the same temperature in both steps, includingperforming both steps simultaneously in one reactor.

[0039] Contrary to chemical conversion of galactose into tagatose theprocess can be run at a pH value optimal for sugars. This significantlyreduces the production of sugar degradation products. As a result,recovery and economy are improved. A typical pH value is about 7.0.Usable pH values and other reaction parameters are i.a. found in U.S.Pat. No. 6,057,135. Yamanaka, K and Wood, W. A. (1966) Methods inEnzymology 9: 596-602, list a number of lactic acid bacteria providingan L-arabinose isomerase enzyme capable of producing ketoses from i.a.D-galactose.

[0040] The product of the invention in the form of syrup is so pure thatit is possible to use tagatose syrups directly. Hitherto it has beennecessary to purify the product, for example to crystallize the impuresyrup and dissolve or solubilize it again.

[0041] As mentioned above, D-Tagatose can be manufactured from alactose-containing source (e.g. cheese whey, casein whey or milk).Lactose is hydrolyzed to equal amounts of galactose and glucose by thelactase, which can be immobilized lactase.

[0042] Galactose is preferably separated from glucose by chromatography.Non-hydrolyzed lactose may be separated and recycled to the enzymecolumn. Galactose is isomerized to tagatose by optionally immobilizedL-arabinose isomerase. Non-isomerized galactose may be separated bychromatography and recycled. The fraction containing D-Tagatose iscrystallized. The crystals are separated from the mother liquor anddried. The mother liquor high in tagatose may berecycled/recrystallized. It is also possible to directly use thetagatose produced as syrup in food products for humans or otherpurposes.

[0043] Thus, there has now been found an effective enzymatic procedurecombined with chromatography for manufacturing of tagatose in high yieldand in a very pure form in one or two reactors. The process has specialadvantages if performed with thermophilic/extremophilic enzymes.

[0044] The new process for the production of tagatose is highlyeffective and clean due to a specific enzymatic conversion combined withrecycling of non-converted products. The process is extremely effectiveand environmentally friendly.

EXAMPLES Example 1

[0045] Hydrolysis of Lactose with Recirculation

[0046] Isomerization of Galactose with Recirculation

[0047] 1. Lactose is produced from whey by ultrafiltration followed bycrystallization.

[0048] 2. A solution of lactose in water (8-40%DS) is hydrolyzed byimmobilized lactase at high or low temperature (either by enzyme fromAspergillus oryzae or a thermophilic organism).

[0049] 3. Glucose and galactose are separated by chromatography.Depending on concentration of feed it may be necessary to concentratefor instance by evaporation.

[0050] 4. Lactose and possible galactooligosaccharides are recycled tothe column containing immobilized enzyme.

[0051] 5. If the concentration of oligosaccharides in the recycling loopis too high (the hydrolysis is undesirably affected), the system isflushed.

[0052] 6. The fraction containing glucose and galactose is isomerized byimmobilized galactose isomerase (from a thermophilic organism)

[0053] 7. The mixture is concentrated for instance by evaporation

[0054] 8. Tagatose is separated by chromatography

[0055] 9. The tagatose fraction is concentrated and possiblycrystallized

[0056] 10. The glucose fraction might be concentrated for sale as asyrup or it may be further processed

[0057] 11. The Galactose fraction is recycled to the isomerase column

Example 2

[0058] Hydrolysis of Lactose without Recirculation with FollowingIsomerization

[0059] 1. Lactose is produced from whey by ultrafiltration followed bycrystallization.

[0060] 2. A solution of lactose in water (8-40%DS) is hydrolyzed byimmobilized lactase (the enzyme originates from Aspergillus oryzae).

[0061] 3. The mixture containing about 46% of glucose, 46% of galactoseis passed through a column containing immobilized galactose isomerase(from a thermophilic organism). About 30% of galactose is converted intotagatose.

[0062] 4. The product is separated into 3 fractions by concentration andchromatographic separation:

[0063] Fraction 1 contains mainly non-converted galactose. This fractionis recycled to the galactose isomerase column

[0064] Fraction 2 contains mainly tagatose. This fraction isconcentrated for crystallization or it is marketed as syrup.

[0065] Fraction 3 contains mainly glucose, but alsogalactooligosaccharides produced by the lactase enzyme as well asun-converted lactose. This fraction is concentrated for sale as syrup orfor further processing, such as crystallization or drying.

Example 3

[0066] Hydrolysis and Isomerization in one Reactor

[0067] 1. Lactose is produced from whey by ultrafiltration followed bycrystallization.

[0068] 2. A solution of lactose in water is passed through a columncontaining immobilized lactase and L-arabinose isomerase (both enzymesoriginating from thermophilic organisms).

[0069] 3. The product is separated into 3 fractions by concentration andchromatographic separation:

[0070] Fraction 1 contains mainly non-converted galactose. This fractionis recycled to the column for enzymatic conversion.

[0071] Fraction 2 contains mainly tagatose. This fraction isconcentrated for crystallization or it is marketed as a syrup.

[0072] Fraction 3 contains mainly glucose, but alsogalactooligosaccharides produced by the lactase enzyme as well asun-converted lactose. This fraction is concentrated for sale as a syrupor for further processing, such as crystallization or drying

Example 4

[0073] One-Reactor Conversion of Lactose to Tagatose with ImmobilizedLactase and Immobilized Isomerase

[0074] The β-glycosidase encoding gene from Sulfolobus solfataricus(Moracci M, Ciaramella M, and Rossi M. [2001] Methods in Enzymology vol.330, p. 201-15) was cloned and expressed in E. coli. The gene wasisolated by polymerase chain reaction (PCR) using purified chromosomalDNA from Sulfolobus solfataricus strain P2. Primers containingadditional restriction sites for NdeI and BamHI were designed to yieldthe entire coding sequence on a fragment which was subsequently clonedinto the standard expression plasmid pET3a (Novagen).

[0075]E. coli cells expressing the enzyme were cultivated, harvested bycentrifugation, lysed in a French pressure cell and cross-linked withglutaraldehyde and polyethylenimine as described in U.S. Pat. No.4,354,105. The immobilized enzyme was recovered by centrifugation andlyophilisation of the pellet. The activity of the immobilized lactasewas 1500 units/g dry weight. One unit was defined as the amount ofenzyme liberating one micromole of glucose per min at 65° C., pH 7, in a30-% (w/v) solution of lactose.

[0076] The L-arabinose isomerase gene from Thermoanaerobacter mathraniiwas cloned and expressed in E. coli as described in patent applicationno. U.S. Ser. No. 09/905,108 (Biotechnological Institute, Denmark).

[0077]E. coli cells expressing the enzyme were cultivated, harvested bycentrifugation, lysed in a French pressure cell and cross-linked withglutaraldehyde and polyethylenimine as described in U.S. Pat. No.4,354,105. The immobilized enzyme was recovered by centrifugation andlyophilisation of the pellet. The activity of the immobilizedL-arabinose isomerase was 50 units/g dry weight. One unit was defined asthe amount of enzyme producing one micromole of D-tagatose per min at65° C., pH 7, in a 30-% (w/v) solution of D-galactose.

[0078] One-milliliter assay mixtures containing 20 mg of immobilizedlactase, 80 mg of immobilized isomerase, 0.30 g of lactose (30%, 875mM), 25 mM K-maleate buffer, pH 6.9, and 5 mM MnCl₂ were incubated at65° C. A control sample without enzymes was treated similarly.Periodically, samples were taken and the concentrations of glucose,galactose and tagatose were determined by high pressure liquidchromatography using an Aminex HPX-87C column (Bio-Rad) and a refractiveindex detector (Waters 410). The mobile phase was de-ionized, degassedwater, the column temperature was 85° C., and the flow rate was 0.6ml/min.

[0079] As shown in FIG. 4, the concentration of glucose increased toabout 800 mM over 24 h, indicating that almost all lactose washydrolyzed to galactose and glucose. The concentration of tagatoseincreased linearly to about 300 mM over 24 h, indicating a bioconversionof 300 mM/800 mM=38%. TABLE Conversion of lactose to tagatose withimmobilized lacS lactase from S. solfataricus and araA isomerase from T.mathranii incubation galactose tagatose time (h) lactose (mM) glucose(mM) (mM) (mM) 0 816 0 0 0 2 176 57 30 4 281 87 48 6 366 116 72 8 430139 98 24 811 316 295

1. A process for manufacturing of tagatose comprising a) hydrolyzinglactose or a lactose containing starting material to obtain galactoseand glucose b) isomerizing the obtained galactose with a L-arabinoseisomerase, and c) chromatographic separation of products and unconvertedcompounds and recycling of unconverted compounds to the process.
 2. Aprocess of claim 1, wherein unconverted lactose is separated bychromatography and recycled to step a) for hydrolysis.
 3. A process ofclaim 1 or 2, wherein unconverted galactose is separated bychromatography and recycled to step b) for isomerization.
 4. A processof any of the preceding claims, wherein step a) and step b) areperformed in one reactor.
 5. A process of any of the preceding claims,wherein the galactose is D-galactose and the tagatose is D-tagatose. 6.A process of any of the preceding claims, wherein the L-arabinoseisomerase used in step b) is thermophilic.
 7. A process of any of thepreceding claims, wherein the lactase used in step a) is thermophilic.8. A process of claim 1 or 2 wherein the L-arabinose isomerase used instep b) is derived from the group consisting of but not restricted toBacillus, Sulfolobus, Thermoanaerobacter and Thermotoga.
 9. A process ofclaim 8, wherein the L-arabinose isomerase is derived fromThermoanaerobacter mathranii.
 10. A process of claim 9 wherein theL-arabinose isomerase is derived from Thermoanaerobacter mathranii DSM11426.
 11. A process of any of the preceding claims, wherein the lactaseused in step a) is derived from the group consisting of but notrestricted to Bacillus, Sulfolobus, Thermoanaerobacter, Thermus andPyrococcus.
 12. A process of claim 1, wherein one or more of the usedenzymes is immobilized.
 13. A process of any of the preceding claims,wherein the temperature used in step(s) a) and/or b) is/are 40-90° C.14. A process of claim 13, wherein the temperature is 60-90° C.
 15. Aprocess of claim 14, wherein the temperature is 60-80° C.
 16. A processof claim 15, wherein the temperature is 65-70° C.
 17. A process of claim16, wherein the temperature is 65° C.
 18. A process according to claim4, wherein the L-arabinose isomerase used is derived fromThermoanaerobacter mathranii and the lactase used is derived fromSulfolobus solfataricus.